Wild-type Cells Research Articles

Glycocalyx Interactions Modulate the Cellular Uptake of Albumin-Coated Nanoparticles.

Albumin-based nanoparticles (ABNPs) represent promising drug carriers in nanomedicine due to their versatility and biocompatibility, but optimizing their effectiveness in drug delivery requires understanding their interactions with and uptake by cells. Notably, albumin interacts with the cellular glycocalyx, a phenomenon particularly studied in endothelial cells. This observation suggests that the glycocalyx could modulate ABNP uptake and therapeutic efficacy, although this possibility remains unrecognized. In this study, we elucidate the critical role of the glycocalyx in the cellular uptake of a model ABNP system consisting of silica nanoparticles (NPs) coated with native, cationic, and anionic albumin variants (BSA, BSA+, and BSA-). Using various methodologies-including fluorescence anisotropy, dynamic light scattering, microscale thermophoresis, surface plasmon resonance spectroscopy, and computer simulations─we found that both BSA and BSA+, but not BSA-, interact with heparin, a model glycosaminoglycan (GAG). To explore the influence of albumin-GAG interactions on NP uptake, we performed comparative uptake studies in wild-type and GAG-mutated Chinese hamster ovary cells (CHO), along with complementary approaches such as enzymatic GAG cleavage in wild-type cells, chemical inhibition, and competition assays with exogenous heparin. We found that the glycocalyx enhances the cell uptake of NPs coated with BSA and BSA+, while serving as a barrier to the uptake of NPs coated with BSA-. Furthermore, we showed that harnessing albumin-GAG interactions increases cancer cell death induced by paclitaxel-loaded albumin-coated NPs. These findings underscore the importance of albumin-glycocalyx interactions in the rational design and optimization of albumin-based drug delivery systems.

The Investigation of Hsp90C-Terminal Inhibitors Containing Amide Bioisosteres.

Heat Shock Protein 90 (Hsp90) is responsible for the proper folding and maturation of ~400 client protein substrates, many of which are directly associated with the ten hallmarks of cancer. Hsp90 is a great target for cancer therapy including melanoma, since Hsp90 inhibition can disrupt multiple oncogenic pathways simultaneously. In this study, we report the synthesis and anti-proliferative activity manifested by a series of Hsp90 C-terminal inhibitors against mutant BRAF and wild-type BRAF melanoma cells. Furthermore, we explored structure-activity relationships (SAR) for the amide moiety of 6 (B1), a novel Hsp90C-terminal inhibitor via introduction of amide bioisosteres. Compound 6 displayed an IC50 of 1.01 μM, 0.782 μM, 0.607 μM and 1.413 μM against SKMel173, SKMel103, SKMel19 and A375 cells, respectively.

MiR-144/451 attenuates lipopolysaccharide-induced lung inflammation by downregulating Rac1 and STAT-3 in macrophages.

MicroRNAs have been shown to play a critical role in lung inflammatory diseases. Here, we report that knocking out miR-144/451 in mice exacerbates lipopolysaccharide (LPS)-induced lung inflammation. The lung inflammation in mice was induced by intratracheal instillation of LPS. Loss-of-function experiments demonstrated that miR-144/451 gene knockout (KO) increased LPS-induced lung inflammation and oxidant stress compared with wild-type (WT) mice, as manifested by increased total bronchoalveolar lavage fluid cells and neutrophil counts, elevated TNF-α and IL-6 levels in bronchoalveolar lavage fluid, enhanced myeloperoxidase activity, and reduced catalase and glutathione peroxidase activity in lung tissues. We also found that LPS significantly decreased miR-451 expression in lung tissues and macrophages; while miR-451 overexpression in LPS-induced RAW264.7 cells remarkably reduced TNF-α and IL-6 levels as well as reactive oxygen species (ROS) production, suggesting a feedback loop might exist in inflammatory cells. Rac1 mRNA and protein levels were downregulated in miR-451-overexpressed RAW264.7 cells. Ex vivo stimulation experiments, performed using alveolar macrophages isolated from miR-144/451 KO mice, confirmed that Rac1 inhibitor alleviated levels of TNF-α and ROS in response to LPS stimulation compared with WT controls. Luciferase reporter assay demonstrated that STAT-3 is a direct target of miR-451. STAT-3 protein levels were elevated in miR-144/451 KO macrophages. LPS treatment also resulted in higher phosphorylation levels of STAT-3 in macrophages from KO mice than in WT cells. Our study identified miR-144/451 as an anti-inflammatory factor in LPS-induced lung inflammation that acts by downregulating Rac1 and STAT-3.

PI 3-kinase/AKT/FOXO1 signaling in smooth muscle cells protects against abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs). Growth factors induce proliferation and cell survival of SMCs. PI 3-kinase (PI3K) is an important downstream mediator in growth factor signaling. Objective This project is based on the hypothesis that PI3K-mediated SMC proliferation can compensate for the loss of SMCs in AAA and thus inhibit AAA progression. Therefore, we determined effects of reduced PI3K activity in SMCs on AAA progression, cell proliferation as well as vascular remodeling. Methods To investigate the role of PI3K, we analyzed mice and aortic SMCs harboring a smooth muscle-specific deficiency of the PI3K catalytic subunit p110α (SM-p110α-/-). AAA development in SM-p110α-/- mice and wild-type (WT) littermates was induced by perfusion of the infrarenal aortic segment with porcine pancreatic elastase (PPE). AAA diameter was determined by echocardiography. Structural changes in the aorta were identified by (immuno)histochemical staining. SMC proliferation was measured in vitro by BrdU incorporation or real-time cell analysis. p110α induced signal transduction pathways were characterized by Western blot and transcriptome analysis. Results PPE treatment led to a massive loss of SMCs in the compromised aortic segment after 3 days in both SM-p110α-/- and WT mice. The aortic diameter was enlarged after 28 days in all PPE-treated mice. However, the increase was significantly higher in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10) (p<0.05). A similar effect was obtained in aged (untreated) mice: In 24-month-old SM-p110α-/- mice, abdominal aortic lumen diameter was increased by 42% compared to 3-month-old animals, but only by 15% in corresponding WT mice. PPE-treated SM-p110α-/- mice were characterized by reduced vascular remodeling with less PCNA-positive proliferating cells compared to WT animals. In addition, proliferation of aortic p110α-/- SMCs in serum-containing medium was impaired. Mechanistic analyses showed that PDGF- and insulin-induced activation of AKT as well as AKT-dependent phosphorylation and inactivation of the transcription factors FOXO-1, -3 and -4 were significantly reduced in p110α-/- SMCs compared to WT cells. Further analyses revealed that specific inhibition of FOXO1 by AS1842856 rescued PDGF or serum stimulated proliferation of p110α-/- SMCs. In addition, expression analysis of FOXOs in patients revealed significantly increased FOXO1 expression in AAA compared to aortic samples from healthy individuals. Conclusion Deficiency of the catalytic PI3K isoform p110α in SMCs promotes the development and progression of AAA. p110α/AKT-dependent inactivation of FOXO1 induces SMC proliferation, which can ameliorate aortic wall damage. As FOXO1 inhibitors and a recently developed p110α activator are available, the PI3K/AKT/FOXO1 signaling cascade may provide a therapeutic target to influence the stability of the aortic wall in AAA.

The prominent pervasive oncogenic role and tissue specific permissiveness of RAS gene mutations

In cancer research, RAS biology has been focused on only a handful of tumor types. While RAS genes have long been suspected as common contributors to a wide spectrum of cancer types, robust evidence is required to firmly establish their critical oncogenic significance. We present a data mining study using DepMap genome-wide CRISPR screening data, which provide substantial evidence to support the prominent pervasive oncogenic role and tissue-specific permissiveness of RAS gene mutations. Differential analysis of CRISPR effect scores identifies K- or N-RAS genes as the most differential gene in contrasts of (K-, N-, combined) RAS mutant versus wild-type cell lines across multiple tissue types. The distinguished tissue-specific pattern of KRAS vs. NRAS as top differential genes in subsets of tissue types and evidence from genome data supported the idea of KRAS- and NRAS-engaged tissue types. To our knowledge, this is the first report of prominent pervasive oncogenic role of RAS mutations revealed by gene dependency data that is beyond the current understanding of the oncogenic role of RAS genes and their well-known involved tissue types. Our findings strongly support RAS mutations as primary oncogenic drivers beyond traditionally recognized cancer types and offer insights into their tissue-specific permissiveness.

The response to single-gene duplication implicates translation as a key vulnerability in aneuploid yeast.

Aneuploidy produces myriad consequences in health and disease, yet models of the deleterious effects of chromosome amplification are still widely debated. To distinguish the molecular determinants of aneuploidy stress, we measured the effects of duplicating individual genes in cells with different chromosome duplications, in wild-type cells (SSD1+) and cells sensitized to aneuploidy by deletion of RNA-binding protein Ssd1 (ssd1Δ). We identified gene duplications that are nearly neutral in wild-type euploid cells but significantly deleterious in euploids lacking SSD1 or in SSD1+ aneuploid cells with different chromosome duplications. Several of the most deleterious genes are linked to translation. In contrast, duplication of other genes benefits multiple ssd1Δ aneuploids over controls, and this group is enriched for translational effectors. Furthermore, both wild-type and especially ssd1Δ aneuploids with different chromosome amplifications show increased sensitivity to translational inhibitor nourseothricin. We used comparative modeling of aneuploid growth defects, based on the cumulative fitness costs measured for single-gene duplication. Our results present a model in which the deleterious effects of aneuploidy emerge from an interaction between the cumulative burden of many amplified genes on a chromosome and a subset of duplicated genes that become toxic in that context. These findings provide a perspective on the dual impact of individual genes and overall genomic burden, offering new avenues for understanding aneuploidy and its cellular consequences.

Introduction of asingle-nucleotide variant, rs16851030, into the ADORA1 gene increased cellular susceptibility to hypoxia.

Aim: Rs16851030, a single-nucleotide variant located in the 3'-untranslated region of the ADORA1 gene, has been proposed as a potential marker of caffeine sensitivity in apnea of prematurity. Besides, it is associated with aspirin-induced asthma and the development of acute chest syndrome. However, its functional significance is still unconfirmed. This study aimed to elucidate the functional impact of rs16851030 by using CRISPR/Cas9 approach to induce the DNA variant and attendant physiological changes.Methods: Rs16851030 was introduced into HEK293 cells via homology-directed repair (HDR). Edited cells were fluorescence-enriched, sorted, isolated, and expanded into single-cell-derived clones. The edit was confirmed by Sanger sequencing. RNA sequencing was used to analyze affected pathways.Results: Rs16851030-mutant cells showed increased susceptibility to hypoxia, a condition related to apnea of prematurity. After 24h of hypoxia, the viability of mutant clones 1 and 2 was low compared with wild-type cells (75.45% and 74.47% vs. 96.34%). RNA sequencing revealed transcriptomic changes linked to this increased vulnerability.Conclusion: Rs16851030 impairs cellular resistance to hypoxia, suggesting its role in conditions like apnea of prematurity. Further research should investigate the molecular mechanisms and transcriptomic alterations caused by rs16851030 under hypoxic conditions.

TRAF4 promotes lung cancer development by activating tyrosine kinase of EGFR

Objective: To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion. Methods: Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay. Results: The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin (r2: 0.438, 0.695, and 0.736, respectively, all P＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P=0.034). Traf4-/- cells had a weakened proliferative capacity than traf4+/+ cells and formed tumors with smaller size (P＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4-/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4+/+ cells [(62.3±10.3)%, P=0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4-/- cells were lower than those of the traf4+/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions: TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

Morphometric Analysis of Actin Networks.

The organization of cytoskeletal elements is pivotal for coordinating intracellular transport in eukaryotic cells. Several quantitative measures based on image analysis have been proposed to characterize morphometric features of fluorescently labeled actin networks. While helpful in detecting differences in actin organization between treatments or genotypes, the accuracy of these measures could not be rigorously assessed due to a lack of ground-truth data to which they could be compared. To overcome this limitation, we utilized coarse-grained computer simulations of actin filaments and crosslinkers to generate synthetic actin networks with varying levels of bundling. We converted the simulated networks into pseudo-fluorescence images similar to images obtained using confocal microscopy. Using both published and novel analysis procedures, we extracted a series of morphometric parameters and benchmarked them against analogous measures based on the ground-truth actin configurations. Our analysis revealed a set of parameters that reliably reports on actin network density, orientation, ordering, and bundling. Application of these morphometric parameters to root epidermal cells of Arabidopsis thaliana revealed subtle changes in network organization between wild-type and mutant cells. This work provides robust measures that can be used to quantify features of actin networks and characterize changes in actin organization for different experimental conditions.

Metabolic Response to Small Molecule Therapy in Colorectal Cancer Tracked with Raman Spectroscopy and Metabolomics.

Despite numerous screening tools for colorectal cancer (CRC), 25 % of patients are diagnosed with advanced disease. Novel diagnostic technologies that are early, accurate, and rapid are imperative to assess the therapeutic efficacy of clinical drugs and identify new biomarkers of treatment response. Here Raman spectroscopy (RS) was used to track metabolic reprogramming in KRAS-mutant HCT116 and SW837 cells, and KRAS wild-type CC cells. RS combined with multivariate analysis methods distinguished nonresponsive, partially responsive, and responsive cells treated with cetuximab, a monoclonal antibody for EGFR inhibition, sotorasib, a clinically approved KRAS inhibitor, and various doses of trametinib, an inhibitor of the MAPK pathway. Cells treated with a combination of subtoxic doses of trametinib and BKM120, an inhibitor of the PI3K pathway, showed a synergistic response between the two pathways. Using a supervised machine learning regression model, we established a scoring methodology trained to a priori predict therapeutic response to new treatment combinations. RS metabolites were verified with mass spectrometry, and enrichment pathways were identified, including amino acid, purine, and nicotinate and nicotinamide metabolism that differentiated monotherapy from combination therapy. Our approach may ultimately be applicable to patient-derived primary cells and cultures of patient tumors to predict effective drugs for individualized care.

Identifying the amino acid domains of ORF59 responsible for interactions with ORF57 and PAN RNA during KSHV lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor, ORF59, is a lytic protein essential for viral DNA synthesis as part of the core replication complex. The multifunctional nature of ORF59 has prompted the investigation into its various functional domains. Prior studies of ORF59 have identified dimerization, DNA interaction, and polymerase interaction domains. The regions of ORF59 responsible for the interaction with the viral mRNA transport accumulation protein (MTA/ORF57) and the viral long non-coding polyadenylated nuclear (PAN) RNA have not been explored. Using a series of previously characterized ORF59 deletion KSHV BACmid mutants, we identified the domains of ORF59 that interact with ORF57 and PAN RNA. Interestingly, amino acids 51-100 were essential for interacting with both ORF57 and PAN RNA. Using this information, we generated a plasmid that expresses a DsRed-tagged polypeptide spanning amino acids 30-100 of ORF59. When the 30-100 aa DsRed-tagged polypeptide expression plasmid was transfected into KSHV wild-type iSLK cells prior to lytic reactivation, a dominant-negative inhibition of virus replication was observed, resulting in a decrease of infectious virus production. Our data suggest that interactions between ORF59 with ORF57 and PAN RNA are important to successful lytic replication.IMPORTANCETo better understand the Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor ORF59, we investigated the interaction of ORF59 with ORF57 and polyadenylated nuclear (PAN) RNA. We used a previously characterized KSHV BACmid containing internal deletions of ORF59 to identify the domains of ORF59 that interact with ORF57 and PAN RNA. Our study revealed multiple domains of ORF59 that are essential for its association with PAN RNA. These domains span amino acids 51-100, 251-300, and 351-396. Additional experiments confirmed amino acids 51-100 are critical for the interaction between ORF59 and ORF57. Using this information, we generated an expression plasmid encompassing the ORF57 and PAN RNA interaction domains of ORF59. The ORF59 polypeptide expression plasmid of amino acids 30-100 functioned as a dominant negative inhibitor during viral reactivation and caused a decrease in virus production. These findings provide valuable insights into the key domains of ORF59, essential for its functionality, and ultimately the production of infectious viruses.

PBP4 is required for serum-induced cell wall thickening and antibiotic tolerance in Staphylococcus aureus.

The bacterial pathogen Staphylococcus aureus responds to the host environment by synthesizing a thick peptidoglycan cell wall, which protects the bacterium from membrane-targeting antimicrobials and the immune response. However, the proteins required for this response were previously unknown. Here, we demonstrate by three independent approaches that the penicillin-binding protein PBP4 is crucial for serum-induced cell wall thickening. First, mutants lacking various non-essential cell wall synthesis enzymes were tested, revealing that a mutant lacking pbp4 was unable to generate a thick cell wall in serum. This resulted in reduced serum-induced tolerance of the pbp4 mutant toward the last resort antibiotic daptomycin relative to wild-type cells. Second, we found that serum-induced cell wall thickening occurred in each of a panel of 134 clinical bacteremia isolates, except for one strain with a naturally occurring mutation that results in an S140R substitution in the active site of PBP4. Finally, inhibition of PBP4 with cefoxitin prevented serum-induced cell wall thickening and the resulting antibiotic tolerance in the USA300 strain and clinical MRSA isolates. Together, this provides a rationale for combining daptomycin with cefoxitin, a PBP4 inhibitor, to potentially improve treatment outcomes for patients with invasive MRSA infections.

Gpr55 deficiency crucially alters cardiomyocyte homeostasis and counteracts angiotensin II induced maladaption in female mice.

Cannabis stimulates several G-protein-coupled-receptors and causes bradycardia and hypotension upon sustained consumption. Moreover, in vitro studies suggest an interference of cannabinoid-signalling with cardiomyocyte contractility and hypertrophy. We aimed at revealing a functional contribution of the cannabinoid-sensitive receptor GPR55 to cardiomyocyte homeostasis and neurohumorally induced hypertrophy in vivo. Gpr55-/- and wild-type (WT) mice were characterized after 28-day angiotensin II (AngII; 1·μg·kg-1min-1) or vehicle infusion. In isolated adult Gpr55-/- and WT cardiomyocytes, mitochondrial function was assessed under naïve conditions, while cytosolic Ca2+ handling was additionally determined following application of the selective GPR55 antagonist CID16020046. Gpr55 deficiency did not affect angiotensin II (AngII) mediated hypertrophic growth, yet, especially in females, it alleviated maladaptive pro-hypertrophic and -inflammatory gene expression and improved inotropy and adrenergic responsiveness compared to WT. In-depth analyses implied increased cytosolic Ca2+ concentrations and transient amplitudes, and accelerated sarcomere contraction kinetics in Gpr55-/- myocytes, which could be mimicked by GPR55 blockade with CID16020046 in female WT cells. Moreover, Gpr55 deficiency up-regulated factors involved in glucose and fatty acid transport independent of the AngII challenge, accelerated basal mitochondrial respiration and reduced basal protein kinase (PK) A, G and C activity and phospholemman (PLM) phosphorylation. Our study suggests GPR55 as crucial regulator of cardiomyocyte hypertrophy and homeostasis presumably by regulating PKC/PKA-PLM and PKG signalling, and identifies the receptor as potential target to counteract maladaptation, adrenergic desensitization and metabolic shifts as unfavourable features of the hypertrophied heart in females.

Stochastic Boolean model of normal and aberrant cell cycles in budding yeast

The cell cycle of budding yeast is governed by an intricate protein regulatory network whose dysregulation can lead to lethal mistakes or aberrant cell division cycles. In this work, we model this network in a Boolean framework for stochastic simulations. Our model is sufficiently detailed to account for the phenotypes of 40 mutant yeast strains (83% of the experimentally characterized strains that we simulated) and also to simulate an endoreplicating strain (multiple rounds of DNA synthesis without mitosis) and a strain that exhibits ‘Cdc14 endocycles’ (periodic transitions between metaphase and anaphase). Because our model successfully replicates the observed properties of both wild-type yeast cells and many mutant strains, it provides a reasonable, validated starting point for more comprehensive stochastic-Boolean models of cell cycle controls. Such models may provide a better understanding of cell cycle anomalies in budding yeast and ultimately in mammalian cells.

Advanced deep-tissue imaging and manipulation enabled by biliverdin reductase knockout.

We developed near-infrared (NIR) photoacoustic and fluorescence probes, as well as optogenetic tools from bacteriophytochromes, and enhanced their performance using biliverdin reductase-A knock-out model (Blvra-/-). Blvra-/- elevates endogenous heme-derived biliverdin chromophore for bacteriophytochrome-derived NIR constructs. Consequently, light-controlled transcription with IsPadC-based optogenetic tool improved up to 25-fold compared to wild-type cells, with 100-fold activation in Blvra-/- neurons. In vivo , light-induced insulin production in Blvra-/- reduced blood glucose in diabetes by ∼60%, indicating high potential for optogenetic therapy. Using 3D photoacoustic, ultrasound, and two-photon fluorescence imaging, we overcame depth limitations of recording NIR probes. We achieved simultaneous photoacoustic imaging of DrBphP in neurons and super-resolution ultrasound localization microscopy of blood vessels ∼7 mm deep in the brain, with intact scalp and skull. Two-photon microscopy provided cell-level resolution of miRFP720-expressing neurons ∼2.2 mm deep. Blvra-/- significantly enhances efficacy of biliverdin-dependent NIR systems, making it promising platform for interrogation and manipulation of biological processes.

Abstract B011: MTA-cooperative PRMT5 inhibition (BMS-986504) induces MTAP del tumor cell-intrinsic immune evasion and regulation

Abstract Homozygous deletion of MTAP gene (MTAP del) occurs in ∼10% of all cancers. BMS- 986504 is a second-generation MTA-cooperative PRMT5 inhibitor designed to preferentially bind to the PRMT5-MTA complex which accumulates in MTAP del cancer cells and has demonstrated clinical proof-of-concept (POC) for selectively inhibiting PRMT5 activity in MTAP del patients as monotherapy. While immunotherapy (IO) has increased patient survival in many cancers and is now the standard of care in first and later lines, most patients ultimately relapse. Thus, there remains an unmet need to improve response for these patients receiving IO. BMS-986504 in combination with anti- PD1 has the potential to increase efficacy, but there is a limited understanding of the interaction between MTAP/PRMT5 biology and PD-1 blockade. Here, we evaluated the BMS-986504-induced immunological and cell killing effects on MTAP del tumor cells and T cells to understand potential mechanisms to support BMS-986504 combination opportunities with anti-PD1 therapy. Using a panel of human PBMC-derived T cells, we first demonstrate that non-activated T cells and T cells activated via anti-CD3/CD28 are insensitive to BMS-986504-induced cell killing at in vitro concentrations equivalent to clinical exposure, supporting the significantly improved selectivity margin of BMS- 986504 relative to first-generation PRMT5 inhibitors that showed T cell toxicity in vitro and on-target dose-limiting hematological toxicity in the clinic. Since previous studies have suggested that MTAP wild-type cells may be affected by MTA secreted from surrounding MTAP del tumors, we assessed T cell viability in the presence of MTA. We found that inactivated T cells maintain insensitivity to BMS-986504 treatment with or without extracellular MTA, but T cells activated via anti-CD3/CD28 are partially sensitive to BMS-986504-induced killing in the presence of MTA only. Using flow cytometry, we characterized the surface expression of immune-related markers on tumor cells treated with BMS-986504 and identified that BMS-986504 dose-dependently increases PD-L1 expression on MTAP del tumor cells, suggesting a potential immune evasion mechanism. Next, using human PBMC-derived T cell and tumor cell co-culture assays, we evaluated the ability of BMS-986504 and anti-PD1 combination to mediate both tumor-intrinsic killing and anti-tumor immune activation in MTAP del cancer cell lines. Taken together, we provide translational proof-of-concept data demonstrating that BMS- 986504 induces tumor cell-intrinsic immunological effects in MTAP del tumors, while having limited impact on T cell viability. This data supports the rationale for BMS- 986504 and IO combination, which may potentially lead to anti-tumor immune activation and improved outcome for MTAP del patients. Citation Format: Josephine Hai, Stephanie Xavier, Sangeeth George, Lars Engstrom, Pete Olson, Tyler Simpson, Ming Lei, Benjamin Chen. MTA-cooperative PRMT5 inhibition (BMS-986504) induces MTAP del tumor cell-intrinsic immune evasion and regulation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B011.

Short-range C-signaling restricts cheating behavior during Myxococcus xanthus development.

Bacteria communicate using both long- and short-range signals. Signaling affects community composition, structure, and function. Adherent communities called biofilms impact medicine, agriculture, industry, and the environment. To facilitate the manipulation of biofilms for societal benefits, a better understanding of short-range signaling is necessary. We investigated the susceptibility of short-range C-signaling to cheating during Myxococcus xanthus biofilm development. A mutant deficient in C-signaling fails to form mounds containing spores (i.e., fruiting bodies) but cheats on C-signaling by wild type in starved cell mixtures and forms spores disproportionately. We found that cheating requires sufficient wild-type cells in the initial mix and can occur both before mound formation and later during the sporulation stage of development. By restricting cheating behavior, short-range C-signaling may have been favored evolutionarily rather than long-range diffusible signaling. Cheating restrictions imposed by short-range signaling may have likewise driven the evolution of multicellularity broadly.

Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria. Conjugative relaxases are translocated through a type IV secretion system into the recipient cell, covalently attached to the transferred DNA strand. We fused relaxase R388-TrwC with the endonuclease Cas12a and confirmed that it can be transported through a T4SS. The fusion protein maintained its activity upon translocation by conjugation into the recipient cell, as evidenced by the induction of the SOS signal resulting from DNA breaks produced by the endonuclease in the recipient cell, and the detection of mutations at the target position. We further show how a template DNA provided on the transferred DNA can be used to introduce specific mutations. The guide RNA can also be encoded by the transferred DNA, enabling its production in the recipient cells where it can form a complex with the Cas nuclease transferred as a protein. This self-contained setup enables to target wild-type bacterial cells. Finally, we extended this strategy to the delivery of relaxases fused to base editors. Using TrwC and MobA relaxases as drivers, we achieved precise editing of transconjugants. Thus, conjugation provides a delivery system for Cas-derived editing tools, bypassing the need to deliver and express a cas gene in the target cells.

IDENTIFYING YB1 AND NPM1 AS PART OF THE MUTANT HISTONE 3 PROTEIN INTERACTOME IN H3 MUTATED PHGG

Abstract AIMS Paediatric high-grade gliomas (pHGG) are the leading cause of childhood cancer deaths, accounting for over 40% of deaths in the UK. pHGG tumours harbour histone H3 mutations, defining 50% of pHGG cases and the major mutations are H3.3K27M and H3.3G34R/V. Our previous proteomic research on H3 intercatomes, identified unique and common interacting binding partners of H3-G34R and H3-G34V mutant proteins relative to wild type H3 protein. Among these binding proteins we have focused on YB-1 and Nucleophosmin 1 (NPM1). YB-1, is multifunctional transcription factor and a well- characterised oncogenic protein. NPM1 is a molecular chaperone and frequently overexpressed in cancers. Our aim is to analyse the role of YB-1 and NPM1 in H3 mutated pHGG, in order to identify targeted therapeutic options for these difficult to treat tumours. METHOD H3 mutated glioma, H3 wild type pHGG and control cell lines were cultured and then subjected to comparative immunofluorescence microscopy, and western blot analyses to investigate YB-1 and NPM1 expression. RESULTS Results showed statistically significant altered protein expression and localisation of YB-1 and NPM1 in H3 mutated pHGG in comparison to wild type and control cells. YB-1 expression was increased in H3 mutated cells and NPM1 showed increased cytoplasmic localization in H3 mutated cells in comparison to H3 WT glioma and control cells. CONCLUSION Results of the study support that YB-1 and NPM1 are important interacting proteins in H3G34-mutated pHGGs and can be explored as potential therapeutic targets in H3 mutated pHGG.

INVESTIGATING GLIOBLASTOMA STEM CELL DIFFERENTIATION IN HUMAN BRAIN 3D ORGANOID CO-CULTURES

Abstract AIMS Glioblastoma (GBM) recurrence is rooted in the ability of residual stem cells to survive treatment and drive relapse. One way to eradicate glioblastoma stem cells (GSCs) is to differentiate them into post-mitotic, non-tumorigenic cell types. Here we aim to differentiate GSCs via genetic manipulation, and test how their response differs in 2D and 3D cultures. METHOD We generated a co-culture model to grow GSC lines with iPSC-derived 3D human brain organoids. Before co-culture, the GSC lines were genetically engineered to overexpress a Doxycycline (Dox) inducible form of the proneural transcription factor ASCL1 (wild type and phosphorylation defective). Live imaging and immunochemistry were performed to analyse tumor composition. RESULTS Our data show that activation of the proneural factor ASCL1 in GSCs when co-cultured with 3D human brain organoids drives reduction in tumor growth and that inhibition of ASCL1 phosphorylation only slightly improves this effect. In comparison to data in 2D cultures, the effect of ASCL1 activation seems enhanced in 3D organoids. At day 7 and 14 of ASCL1 induction, there are only few cancer cells left in the organoids, while, in 2D, wildtype ASCL1-expressing cells continue to grow, even if at a slower rate than control GSCs. CONCLUSION Our data show that cancer stem cells embedded in 3D human brain organoids behave differently than when in 2D culture on plastic and respond more effectively to differentiation treatments. Our model provides a platform to test novel treatments for GBM.